INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2107-2114, 2015

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool Ryo Tsumura1,2, Ryuta Sato1,2, Fumiaki Furuya1, Yoshikatsu Koga1, Yoshiyuki Yamamoto3, Yuki Fujiwara1,2, Masahiro Yasunaga1 and Yasuhiro Matsumura1 1

Division of Developmental Therapeutics, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba 277-8577; 2Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561; 3Department of Gastroenterology and Hepatology, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8576, Japan Received May 27, 2015; Accepted July 6, 2015 DOI: 10.3892/ijo.2015.3210

Abstract. Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen.

Correspondence to: Dr Yasuhiro Matsumura, Division of Developmental Therapeutics, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan E-mail: [email protected]

Abbreviations: MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single photon emission computed tomography; CT, computed tomography; mAb, monoclonal antibody; ADCs, antibody-drug conjugates; TF, tissue factor; SPR, surface Plasmon resonance; PCS, photon correlation spectroscopy; DLS, dynamic light scattering; TBR ratio, tumor-to background ratio; Ka, the association rate constant; Kd, the dissociation rate constant; KD, the binding affinity constant; EPR effect, enhanced permeability and retention effect Key words: tissue factor, in vivo fluorescence imaging, antibody, Fab fragment, pancreatic cancer

In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-AlexaFlour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis. Introduction Of all clinical cancers, pancreatic cancer has one of the poorest prognoses, with an overall 5-year survival rate of ~6% (1). Therefore, detection of the tumor and its micrometastases at an early stage, curative resectability of the tumor, and development of effective drugs for its treatment are desired. In present diagnostics, several non-invasive imaging technologies, such as magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) and computed tomography (CT) are available for clinical use (2). To discriminate the tumor from normal tissues, several monoclonal antibody (mAb)-based probes have been developed by exploiting tumorspecific molecules. MAbs have also been used to treat some types of cancer (e.g., malignant lymphoma, breast cancer) (3,4). Moreover, antibody-drug conjugates (ADCs), by which an anticancer drug as a payload is preferentially delivered to the tumor tissue with minimal adverse effects, are under development. Thus, mAbs with high and specific affinity for tumor antigens are potentially excellent tools for cancer diagnosis and treatment. It is well-known that cancer invasion is accompanied by an activation of blood coagulation (5). Recently, several clinical studies have revealed that cancer patients are at a high risk for the development of thrombosis (6). Hemorrhage from the tumor vessels by the invading tumor cells and subsequent fibrin clot formation to stop the bleeding occur repeatedly

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Tsumura et al: ANTI-TISSUE FACTOR FAB FRAGMENT FOR TUMOR DIAGNOSIS

in tumor tissues (7). Moreover, this state lasts in solid tumor tissues for as long as the tumor cells survive in the body (7). Tissue factor (TF) is a 47-kDa transmembrane glycoprotein that is known to initiate the extrinsic coagulation cascade and to play a critical role in hemostasis. Therefore, it is conceivable that high expression levels of TF may be one of the major causes of the hypercoagulable state in tumors. TF is well known to be expressed at high levels in many types of solid tumors, such as pancreatic cancer, glioma, colorectal cancer, non-small cell lung cancer, ovarian cancer, prostate cancer and breast cancer (8-10). TF is also known to be expressed in the tumor stromal cells (11-13). Some studies have indicated that activation of TF signaling in tumor cells enhances tumor growth, metastasis, inflammation and angiogenesis (14-18). Furthermore, TF expression has been shown to be correlated with a poor prognosis in patients with cancer (19-22). We hypothesized that TF may be a promising target for imaging probes or delivery of anticancer drugs into tumor tissues, e.g., pancreatic tumor tissues. We and other groups have already reported the usefulness of anti-TF mAb in cancer imaging and therapy, including the Fab and scFv fragments (23-29). However, the optimum size as a molecular probe has not been fully evaluated from the perspective of biochemical characteristics and bio-distribution within the tumor tissue. In the present study, we prepared an anti-TF mAb and its Fab fragment, and investigated their biochemical and pharmacological characteristics in vitro and in vivo, in order to determine their suitability for application in the diagnosis and treatment of cancer. Materials and methods Antibodies and cell line. We developed the clone 1849 rat mAb IgG2b which reacts with human TF antigen but not with mouse TF antigen. As an isotype control antibody, we also developed clone 372 rat mAb IgG2b which did not react with human and mouse TF antigens (30). The human pancreatic cancer cell line BxPC3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was maintained in RPMI-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 100 U/ml of penicillin G, 100 µg/ ml of streptomycin, and 0.25 µg/ml of amphotericin B (Wako) in a 5% CO2 atmosphere at 37˚C. Generation of the Fab fragment. The 1849 and control mAbs were digested with papain (Worthington Biochemical, Freehold, NJ, USA) at a protein/enzyme ratio of 250:1, in a reaction buffer containing 100 mM sodium phosphate, 2 mM EDTA, and 10 mM Cysteine-HCl, pH 7.0, for 2 h at 37˚C. The reaction buffer was then changed to 5 mM sodium phosphate buffer (pH7.5) for removing Cysteine-HCl using Amicon Ultra (Merck-Millipore, Darmstadt, Germany). The 1849-Fab and control-Fab were purified in a CHT ceramic hydroxyapatite column using a salt concentration gradient (Bio-Rad, Hercules, CA, USA). Purity of the antibodies. The purity of the antibodies was evaluated by SDS-PAGE and Bioanalyzer analysis (Agilent Technologies, Santa Clara, CA, USA) under non-reducing

conditions. In SDS-PAGE, the samples were loaded (2 µg/lane) on a 4-15% polyacrylamide gradient gel (Bio-Rad). The gel was then stained with Coomassie Brilliant Blue R-250 and scanned with ChemiDoc XRS+ (Bio-Rad). The purity and molecular size of the antibodies were measured using an Agilent bioanalyzer, as prescribed in the manual, using the Agilent Protein 230 kit. Particle size determination and surface plasmon resonance (SPR) analysis. Particle size was determined using DelsaNano HC (Beckman Coulter, Brea, CA, USA) on the basis of photon correlation spectroscopy (PCS) and dynamic light scattering (DLS). Biacore T200 (GE Healthcare, Uppsala, Sweden) was used to assess the binding of the 1849 antibodies to recombinant human TF (rhTF) antigen. Following standard amine chemistry protocols, rhTF antigen was diluted to 1 µg/ml in 10 mM sodium acetate buffer (pH 5.0) and immobilized on the CM5 sensor chips (GE Healthcare) at lower density (17.7 resonance units) to only allow monovalent binding. We used the single-cycle kinetics to measure the affinity of the antibodies. Antibodies in PBS were passed over the sensor chips, and the interactions were monitored for 30 sec. The sensor surface was washed with PBS to detect dissociation and then regenerated with 10 mM Glycine-HCl (pH 1.5) at the end of each experiment. Antibody labeling. The 1849-whole IgG, 1849-Fab, controlwhole IgG and control-Fab were chemically labeled with the fluorescent dyes Alexa-Fluor-647 (AF647, Invitrogen, Eugene, OR, USA), according to the manufacturer's protocol (MP 00143, Amine-Reactive Probes, Invitrogen). Following the labeling reaction, the antibody concentration and degree of labeling were measured by determining the absorbance values at 280 nm and 650 nm with a Nano Drop ND-1000 spectrometer (Thermo Fisher Scientific, Wilmington, DE, USA). The final concentration of the antibodies-AF647 was 1.0 mg/ml, and the final dye molarities of 1849-whole IgG-AF647, 1849-Fab-AF647, control-whole IgG-AF647 and control-Fab-AF647 were 19.5, 18.2, 21.9 and 21.5 µM, respectively. Confocal fluorescence microscopy analysis. BxPC3 cells were planted on BD Falcon 4-well chamber slide at 1x105 cells/well. After 24 h of incubation, cells were incubated with 500 µl of RPMI medium containing 0.1 µM 1849-whole IgG-AF647, 1849-Fab-AF647, control-whole IgG-AF647 or controlFab‑AF647 for 1 h at 37˚C. Cells were washed three times with PBS, then fixed with 4% paraformaldehyde in PBS for 15 min at RT and stained with DAPI solution (1 µg/ml) for 5 min at RT. The slides were covered with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Fluorescence images were obtained with a fluorescence microscope, BIOREVO BZ9000 (Keyence, Osaka, Japan). Flow cytometric analysis. The cell-binding activities of the antibodies-AF647 to BxPC3 cells were evaluated by flow cytometry. Briefly, BxPC3 cells were harvested and suspended in PBS with 0.5% bovine serum albumin (BSA, Wako) and 2 mM EDTA (B.E.PBS) at a density of 4x105 cells/ml. The BxPC3 cells were then incubated with the antibodies-AF647

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Table I. Particle size and binding parameters of antibodies. 1849 whole IgG 1849 Fab Control whole IgG Control Fab

Particle size (nm) KD (M) Ka (1/Ms) Kd (1/s) 13.1±4.6 3.665x10-10 3.770x104 1.382x10-5 5.0±2.4 2.001x10-9 2.895x104 5.795x10-5 13.0±4.8 N/A N/A N/A 5.5±1.8 N/A N/A N/A

The particle size is shown as the mean ± standard deviation. N/A showed no data.

at each concentration (1, 10 and 100 µM) for 30 min on ice, and washed three times with B.E.PBS. Subsequently, propidium iodide (0.4 µl/ml; PI, Invitrogen) was added, and the cell binding activities were analyzed with the guava easyCyte (Millipore) using FlowJo analysis software (Tree Star Inc., Ashland, OR, USA). As a negative control, we added the fraction containing PI and not the antibodies-AF647. Animal models. Four-week-old female BALB/c nude mice were purchased from SLC Japan (Shizuoka, Japan). A week later, the mice were inoculated subcutaneously in the right flank with 1x106 BxPC3 cells suspended in 100 µl PBS. The test antibodies were injected intravenously into the tail vein of the mice when the tumor volume reached ~100-150 mm3, as measured with calipers, and calculated by the formula: volume = length x (width)2 x 1/2. All animal procedures and experiments were conducted with the approval of the Committee for Animal Experimentation of the National Cancer Center, Japan. These guidelines meet the ethical standards required by law and also comply with the guidelines for the use of experimental animals in Japan. In vivo and ex vivo fluorescence imaging. In vivo and ex vivo fluorescence imaging were performed using IVIS Kinetic imaging system and analyzed using IVIS Living Imaging 3.0 software (Caliper Life Sciences, Hopkinton, MA, USA). A filter set (excitation at 605 nm, emission at 640 nm) was used for acquiring the fluorescence of the AF647 conjugated antibodies. All fluorescence images were acquired in identical illumination settings and normalized as photons per second per centimeter square per steradian (p/s/cm2/sr). Quantitative data were obtained from ROI (regions of interest) analysis of the fluorescence images. The mice were injected with ~100 µg of antibodies-AF647 via the tail vein in both studies. In vivo imaging was performed at 1, 2, 3, 6, 9, 12, 24 and 72 h post-injection. The mean fluorescence intensity of the left flank region, on the side contralateral to the tumor, was used as the background value in order to investigate the tumor-to-background ratio (TBR) of 1849-whole IgG-AF647 and 1849-Fab-AF647. The TBR was determined as the mean fluorescence intensity of the tumor divided by the mean background intensity. Statistical analysis were performed using Student's t-test. P-values of